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Title: KiPIK Screening: A Novel Method to Identify Kinases Responsible for Phosphorylation events of Interest.
Authors: Watson, Nikolaus Allan
Issue Date: 2019
Publisher: Newcastle University
Abstract: Protein phosphorylation is one of the most abundant forms of post-translational modification and regulates nearly every aspect of cell biology. Understanding the function of a particular phosphorylation event depends to a great extent on identifying the kinase responsible for catalysing it. However, while advances in mass spectrometry based phosphoproteomics have seen an explosion in the ability to detect phosphorylation events occurring in cells, methodological limitations make identifying the kinase responsible for specific phosphorylation events challenging. This thesis explores this problem, beginning with a discussion of the determinants of kinase-substrate specificity in a cell. This is followed by a review of methodologies currently available for identifying kinases responsible for specific phosphorylation events, and a chapter exploring the utility of one of these techniques (siRNA kinome screening) for identifying kinases required for specific histone phosphorylation events in mitosis. We then report the development of KiPIK screening (Kinase Inhibitor Profiling to Identify Kinases), a novel general method for identifying the kinase responsible for a phosphorylation event of interest. The method exploits the fact that in recent years large numbers of kinase inhibitors have been profiled for inhibitory activity on near-kinome-wide panels of recombinant kinases. The method treats the inhibitory information for each kinase as a ‘fingerprint’ for the identification of kinases acting on target phosphorylation sites in cell extracts. In this thesis we detail the development of the technique and validate it on diverse known kinase-phosphosite pairs, including two mitotic histone phosphorylations carried out by Haspin and Aurora B, EGFR autophosphorylation, and the phosphorylation of integrin 1 by Src-family kinases. Finally, we use it to identify the kinase responsible for an as yet unassigned mitotic phosphosite on the Chromosomal Passenger Complex component INCENP. KiPIK screening is broadly applicable and technically straightforward. In addressing the methodological insufficiency in this fundamental area, it has the potential to benefit research widely.
Description: Ph. D. Thesis
Appears in Collections:Institute for Cell and Molecular Biosciences

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